Angiogenesis, the formation of new capillaries from already existing blood vessels, is a procedure which is essential during development and during physiological conditions that require increased vascularisation, such as wound healing and the menstrual cycle. The resting vasculature is tightly regulated by a balance between pro- and anti-angiogenic factors. This balance is disturbed in a number of pathological processes, resulting in deficient angiogenesis, as in ischemic conditions, or excessive angiogenesis, as in rheumatoid arthritis, diabetic retinopathy and tumour growth. It has lately become well established that many types of tumours need to stimulate infiltration of new capillaries to grow and metastasise. Many interesting new therapies, based on pro- and anti-angiogenic substances, are therefore currently being tested in the clinic (Risau et al (1997), Carmeliet et al (2000), Folkman et al (1995, 2000), Hanahan et al (2000), Kerbel et al (2000)).
Histidine Rich Glycoprotein (HRGP) was identified by Heimburger et al in 1972. The protein is synthesized in the liver and has an unusually high content of Pro and His residues. These residues are predominantly situated in a His/Pro region in the centre of the protein and seem to be critical for its function. Until recently, though, very little has been known about the physiological role of HRGP. For a review, see Heimburger et al (1972), Koide et al (1986).
HRGP is present in human plasma at a concentration of approximately 100 μg/ml, which is considered to be very high. The amino acid sequence of mouse, rat, rabbit and human HRGP have been resolved and the protein seems to be well conserved among these vertebrate species (Drasin et al (1996), Hulett et al (2000), Borza et al (1996), Koide et al (1986)). See nucleotide and amino acid sequences of human HRGP under Genbank accession number NM000412.
After proteolytical cleavage from its 18 amino acid long signal peptide, mature HRGP can be divided into three main regions: The N-terminal region, the His/Pro region and the C-terminal region, all displaying different properties. Furthermore, the three regions are suggested to be responsible for binding different ligands. The N-terminal region contains two cysteine protease inhibitor (cystatin)-like stretches (FIG. 1A), which allows the classification of HRGP as a member of the cystatin superfamily, together with .alpha.2HS glycoprotein, cystatin and kininogen, whereas the His/Pro region is very rich in proline and histidine residues resulting in e.g. the human form containing 12 more or less conserved tandem repeats of the pentapeptide HHPHG (SEQ ID NO: 32). In plasma, both the His/Pro region and the C-terminal region are disulfide bonded to the cystatin-like stretches in the N-terminal region (Borza et al (1996)).
High molecular weight kininogen (HK) is structurally related to HRGP, possibly through a gene duplication, as the genes for HK and HRGP are located in close proximity on chromosome 3q. HK has been shown to interfere with endothelial cell function via a part of the protein with sequence similarities to the His/Pro-rich region of HRGP (Zhang et al (2000)).
In general, HRGP binds a variety of different ligands, which can be divided into three major groups: ligands belonging to the coagulation/fibrinolysis system (e.g. heparin, plasminogen and fibrinogen), small ligands (e.g. heme and transition metal ions) and cells (e.g. T-cells, monocytes/macrophages) (Lamb-Wharton et al (1993), Olsen H M et al (1996)).
Furthermore, HGRP binds extracellular matrix components, such as thrombospondin-1 (TSP-1) and vitronectin. Due to its binding to TSP-1, HRGP has previously been suggested to be proangiogenic, (Lijnen et al (1985)).
Juarez et al. (2002) reported that the His/Pro-rich domain of rabbit HRGP purified from plasma inhibits endothelial cell proliferation and vascularisation of matrigel plugs. Using recombinant HRGP, the data could even be extended to provide evidence for in vivo effects on tumour vascularisation. In a previous paper, HRGP was suggested also, under certain circumstances, to promote angiogenesis and to attenuate the anti-angiogenic effect of TSP-1 by complex-formation between the two proteins (Simantov et al (2001)). Juarez et al. suggested that this reported effect is dependent on contamination of the HRGP preparation by plasminogen, which could affect angiogenesis and TSP-1 indirectly.
Other examples of suggested functions of HRGP are modulation of fibrinogenesis (Kluszynski et al, 1997), inhibition of insoluble immune complex formation (Gorgani, 1997) and, recently, potentiation of the ingestion of apoptotic cells by macrophages (Gorgani et al (2002).
In WO 02/076486, the inventors for the first time describe the use of HRGP polypeptides, or its central regions, for the inhibition of anglogenesis. The application further shows methods for inhibiting angiogenesis by administering such a polypeptide to a mammal. Further disclosed are pharmaceutical compositions and articles of manufacture comprising HRGP polypeptides, antibodies and receptors that bind to an HRGP polypeptide, polynucleotides, vectors and host cells that encode HRGP polypeptides.
Later on, WO 02/064621 discloses a selection of HRGP polypeptides, or subfragments thereof, more particularly, specific, H/P-rich, repetitive pentapeptides from a histidine/proline-rich domain of HRGP, as defined by the invention, as being anti-angiogenic. The subfragments are described as inhibitors of angiogenesis and are to be used for the treatment of diseases or conditions, in which angiogenesis is pathogenic. The compounds are predicted to have anti-tumour activity and are proposed to be of use in methods for inhibiting the growth of primary tumours or metastases. Also postulated are antibodies specific for the His-Pro rich domain of HRGP, as stimulators of angiogenesis for promoting neovascularisation in pertinent disease states.
However, none of the previous findings correctly specifies the actual active region, nor the minimal functional entity of HRGP, both being for the first time disclosed by the present invention. Firstly, the inventors are able to show that a central region of human HRGP, defined as amino acid region 240-390 (as seen in SEQ.ID.NO: 2) of mature human HRGP, appears to exist as an endogenous, naturally occurring polypeptide in the human body. This is the first identification of a naturally occurring smaller protein subfragment of human HRGP, comprising similar features to the mature protein, such as anti-angiogenic activity. The identification of this subfragment was then followed by further successful attempts aiming to identify a minimal active entity of the protein, which still possesses an anti-angiogenic activity similar to the naturally occurring fragment. These attempts in turn generated a presently minimal active entity comprising five amino acids derived from the central region of human HRGP and a selection of other subfragments of the central region of human HRGP, also comprising anti-angiogenic activity.
Having thus access to one or more minimal functional entities, a substantially shorter peptide, such as the minimal active entity previously mentioned, or any other HRGP polypeptide/subfragment disclosed by the present invention, can be used as a medicament. The advantages of using a shorter subfragment of HRGP are many, e.g. administration of long peptides is often associated with difficulties due to instability. Also, synthesis of longer peptides is often problematic, whereas shorter peptides are more convenient to synthesise. What is more, they are often less toxic due to higher specificity, which in praxis leads to less side effects.